This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The type III secretion system (TTSS) is a multi-protein molecular machine that is used by many Gram-negative bacteria to dock onto the eukaryotic host cell membrane and export effector proteins to initiate infections. Its apparatus is composed of a basal body that spans the inner and outer bacterial membranes, an external needle, and a tip protein. The needle is formed by the assembly of approximately 120 copies of needle monomers around a central channel. However, the structures of the N-terminal regions of needle monomers that form the channel are not well defined due to their flexible nature. In this proposal, we will combine computational and experimental approaches to determine the structure of the N-terminal regions of the needle proteins, refine the needle model, and characterize the structural basis of how proteins are transported across this channel. Since the bacterial pathogens that use the TTSS pose major problems in public health and safety due to the diverse diseases they cause and the development of antibiotic resistance, a comprehensive understanding of the effector translocation mechanism used in the TTSS will facilitate vaccine and drug developments against the diseases caused by these bacteria.